Method for breeding tomatoes having reduced water content and product of the method

ABSTRACT

A method for breeding tomato plants that produce tomatoes with reduced fruit water content including the steps of crossing at least one  Lycopersicon esculentum  plant with a  Lycopersicon  spp. to produce hybrid seed, collecting the first generation of hybrid seeds, growing plants from the first generation of hybrid seeds, pollinating the plants of the most recent hybrid generation, collecting the seeds produced by the most recent hybrid generation, growing plants from the seeds of the most recent hybrid generation, allowing plants to remain on the vine past the point of normal ripening, and screening for reduced fruit water content as indicated by extended preservation of the ripe fruit and wrinkling of the fruit skin.

FIELD OF THE INVENTION

The present invention relates to a method for breeding tomatoes havingreduced water content and/or with the trait of drying while stillattached to the vine, and to tomatoes having reduced water content andto products of the method.

BACKGROUND OF THE INVENTION

Dehydrated tomato products comprise an important portion of the tomatoindustry. The production of tomato pastes, ketchup and other processedtomato products is dependent on the energy-requiring steps ofdehydration. The production of “sun-dried” tomato products consists ofdehydrating cut tomato fruit either in the sun or in drying ovens.

Dry matter content of mature tomato fruit can range from approximately5–10% (Davies, J. N. and Hobson, G. E. 1981. The constituents of tomatofruit—the influence of environment, nutrition and genotype. CRC CriticalReviews in Food Sci and Nutr. 15:205–280), depending largely on fruitsize. Generally, processing tomato cultivars produce mature fruit with ahigher water content of approximately approximate 94–95%. Smaller,“cherry”-type tomato fruit, with a fresh weight of 10–20 gramsfrequently have higher concentration of solids (dry weight) and hencereduced water concentrations of approximately 90% (10% dry weight).

Generally, tomato fruit development stages can be classified as thepre-climacteric stage, which is comprised of the early stages of fruitgrowth until incipient ripening, the climacteric stage and thepost-climacteric or senescent stage. Once the fruit is fully ripe,tissue disorganization occurs, with pathogens contributing to the tissuedisorganization, and characteristics associated with “overripening” andsubsequent rotting of the fruit become apparent (Grierson, D. and Kader,A. A. 1986. Fruit ripening and quality. In: Atherton, J. G. and Rudich,J. Eds.: The Tomato Crop. Chapman and Hall, London, pp. 241–280).

The production of raisins from grape berries (Vitis vinifera) is a wellknown process in which the dehydration process occurs by diffusion ofwater through a waxy cuticle (Martin, R. J. L. and Scott, G. L. 1957.The physical factors involved in the drying of Sultana groups.Australian Journal of Agricultural Research. 8:444–459). For whole grapeberries, the drying process is generally assisted by various dippingtreatments of the berry, such as the soda-dip method (Nury, F. S.,Brekke, J. E. and Bolin, H. R. 1973. Fruits. In: Van Arsdel, W. B.,Copley, M. J. and Morgan, A. I., Eds: Food Dehydration. Avi PublishingCo., Westport, Conn. vol. 2, pp. 158–198). In brief, in this method theberries are dipped in a 0.2–0.3% solution of caustic soda (sodiumhydroxide) at a temperature, of about 200° F. for a few seconds and arethen rinsed with cold water before dehydration. The purpose of thedipping is to modify the berry cuticle so that transpiration of watervapor across the cuticle may proceed at a faster rate.

The tomato, like the grape, is botanically classified as a berry and hasa waxy cuticle on the fruit epidermis (Baker, E. A., Bukovac, M. J. andHunt, G. M. 1982. Composition of tomato fruit cuticles as related tofruit growth and development. In: Cutler, D. F., and Alvin, K. L. andPrice, C. E., Eds: The Plant Cuticle. Academic Press, London, pp.33–44). However, tomatoes will generally undergo degradation if theyremain on the vine after ripening. In the case of tomatoes, theharvested fruit is generally cut in half in order to increase thedehydration rate. Alternatively, whole fruit may be pierced in order tofacilitate fluid movement (Ojimelukwe, P. C. 1994. Effects of processingmethods on ascorbic acid retention and sensory characteristics of tomatoproducts. J. Food Sci. Technol. 31:247–248). Drying of the slices ofpierced tomato fruit may take place either in the sun or in variousforms of drying ovens based on non-solar energy input.

There are disadvantages to sun-drying since it depends on weatherconditions and inclement weather leads to losses. Similarly, there aredisadvantages to the use of drying ovens as these are energy consuming.Both sun drying and oven drying may lead to losses in food quality. Forexample, levels of ascorbic acid, one of the major nutritionalcontributions of tomatoes in the human diet, decrease significantly inresponse to sun-drying or oven-drying (Ojimelukwe, P. C. 1994. Effectsof processing methods on ascorbic acid retention and sensorycharacteristics of tomato products. J. Food Sci. Technol. 31:247–248).Furthermore, the necessity to cut the tomato fruit in half before thedrying process does not allow for the production of whole dried tomatofruit.

Wild species of the genus Lycopersicon, such as L. hirsutum, may containwithin their genetic makeup expressed characteristics not generallypresent within the L. esculentum species. These genetic traits may betransferred to the cultivated L. esculentum. For example, the genetictrait of sucrose accumulation is present in mature fruit of the subgenusEriopersicon (including L. hirsutum, L. chmiliewskii and L. peruvianum)and this trait has been transferred to L. esculentum, using classicalgenetic breeding techniques, as well as molecular genetic techniques(Schaffer, A. A., Petreikov, M., Miron, D., Fogelman, M., Spiegelman,M., Bnei-Moshe, Z., Shen, S., Granot, D., Hadas, R., Dai, N., Levin, I.,Bar, M., Friedman, M., Pilowsky, M., Gilboa, N. and Chen, L. 1999.Modification of carbohydrate content in developing tomato fruit.Hortscience 34:12–14). The wild species of Lycopersicon, however, mayalso serve as a source of unexpressed genetic traits that can contributeto the value of cultivated plants (Bernacchi, D., Beck-Bunn, T., Eshed,Y., Lopez, J., Petiard, V., Uhlig, J., Zamir, D. and Tanksley, S. 1998.Advanced backcross QTL analysis in tomato. Identification of QTLs fortraits of agronomic importance from Lycopersicon hirsutum. Theor. Appl.Genet. 97:381–397).

SUMMARY OF THE INVENTION

The present invention seeks to provide a method for breeding tomatoeshaving fruit that naturally dehydrate while still attached to the tomatoplant and thus have a reduced water content, and to tomatoes havingreduced water content and to products of the method.

The development of tomato varieties with the trait of naturallydehydrating while still attached to the vine, without the accompanimentof degradative processes leading to fruit breakdown is highly valuableto the various components of the tomato industry. It can contribute toreduction of processing costs and energy expenditures in the productionof pastes, sauces and ketchups. It can contribute to the production ofhigh quality dried and semi-dried (raisin-type) tomato products. It cancontribute to the improvement of tomato fruit transport since the volumeof transported material will be decreased. It can improve the storageability of the tomato fruit since reduced water content will beaccompanied by increased soluble solids concentration which contributesto the resistance to microbial spoilage.

There is thus provided in accordance with a preferred embodiment of thepresent invention a method for breeding tomato plants that producetomatoes with reduced fruit water content including the steps ofcrossing at least one Lycopersicon esculentum plant with a Lycopersiconspp. to produce hybrid seed, collecting the first generation of hybridseeds, growing plants from the first generation of hybrid seeds,pollinating the plants of the most recent hybrid generation, collectingthe seeds produced by the most recent hybrid generation, growing plantsfrom the seeds of the most recent hybrid generation, allowing plants toremain on the vine past the point of normal ripening, and screening forreduced fruit water content as indicated by extended preservation of theripe fruit and wrinkling of the fruit skin.

In accordance with a preferred embodiment of the present invention thesteps of pollinating, collecting the seeds, and growing plants arerepeated at least once.

Further in accordance with a preferred embodiment of the presentinvention the step of pollinating includes self pollination.

Still further in accordance with a preferred embodiment of the presentinvention the step of pollination includes back crossing with aLycopersicon esculentum plant.

Additionally in accordance with a preferred embodiment of the presentinvention the Lycopersicon spp plant is a Lycopersicon hirsutum plant.

In accordance with a preferred embodiment of the present invention themethod additionally includes the steps of crossing plants derived fromhybrid seeds whose progeny show reduced fruit water content with aLycopersicon plant, growing the crossed plants, and selecting plantswith tomato fruits having an increased dry weight percentage as comparedto fruit from a non-crossed Lycopersicon. The steps of crossing andselecting may be repeated at least once. The crossing may include sexualor asexual crossing. The asexual crossing may include somatic cellhybridization.

Further in accordance with a preferred embodiment of the presentinvention the method additionally includes the step of propagating theplants with tomato fruits having the desired characteristics. The stepof propagating may include vegetative propagation or propagation byseed.

In accordance with a preferred embodiment of the present invention themethod additionally includes the steps of crossing plants derived fromhybrid seeds whose progeny show reduced fruit water content with aLycopersicon plant, growing the crossed plants, harvesting ripe tomatofruits before signs of dehydration thereof, and allowing the fruits todehydrate after removal from the plant.

There is also provided in accordance with a preferred embodiment of thepresent invention a tomato fruit characterized by a capability ofnatural dehydration while on a tomato plant, natural dehydration beingdefined as wrinkling of skin of the tomato fruit when the fruit isallowed to remain on the plant after a normal ripe harvest stage, thenatural dehydration being generally unaccompanied by microbial spoilage.

There is also provided in accordance with a preferred embodiment of thepresent invention a tomato fruit characterized by an untreated skinwhich permits hydration of the fruit so as to obtain wrinkling of theskin, the dehydration being generally unaccompanied by microbialspoilage.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Reference is now made to a method for breeding tomatoes having fruitthat naturally dehydrate while still attached to the tomato plant andreduced water content.

The method for breeding tomato plants includes first hybridizing atleast one Lycopersicon esculentum plant with a wild Lycopersicon spp.plant. The fruits of the L. esculentum plants are then allowed to ripenand the hybrid (F₁) seeds are collected. The collected F₁ seeds are thenplanted and F₁ plants are grown and allowed to self-pollinate. Selfingmay be continued for at least one additional generation or the F1 plantsmay be crossed to a L. esculentum parental plant. Fruits from selfed orbackcrossed generations are allowed to remain on the vine past the pointof normal ripening, as determined by change of fruit color, and screenedfor the presence of natural dehydration. Natural dehydration, or reducedwater content, is indicated by the wrinkling of the fruit skin when thefruit is allowed to remain on the vine after the normal red ripe harveststage.

Plants from any of the selfed generations may be propagated for use byvegetative propagation methods such as micropropagation or by sexualpropagation methods. The plants may also be crossed with other L.esculentum cultivars to create varieties that incorporatecharacteristics other than reduced fruit water content. The varietiesmay then be propagated by vegetative or sexual propagation methods.

Plants from any of the selfed generations may also be back crossed to L.esculentum for at least one generation. The fruits of the last backcross generation are allowed to remain on the vine past the normal pointof ripening. The appearance of dehydration as evidenced by wrinkling ofthe fruit skin indicates reduced water content in the fruit. Plantsselected for this trait may then be propagated either vegetatively or byseed based propagation. Selected plants may then also be crossed withother L. esculentum cultivars to create varieties that incorporatecharacteristics other than reduced fruit water content. The varietiesmay then also be propagated by vegetative or sexual propagation methods.

Reference is now made to the following example that illustrates theinvention.

EXAMPLE 1

Plants of the L. esculentum breeding line 1630 (a Volcani Institute malesterile breeding line, used to simplify the production of theinterspecific hybrid) were pollinated with pollen of the wild species L.hirsutum (LA1777). Hybrid F1 plants were grown and allowed toself-pollinate, generating F2 seed. F2 seed were sown and about 350plants were grown in a screenhouse and allowed to self-pollinate. Ripefruit from each individual plant that produced fruit were individuallyanalyzed for soluble solids (refractometrically) to insure the linesalso included the characteristic of high soluble solids. Only 25 of theinterspecific F2 plants freely produced fruit. Three F2 plants wereselected based on their high sugar content (Brix in excess of 10) whenripe. For example, fruit of F2-82 had 71 mg soluble sugar, composed ofsucrose, glucose and fructose, per gram fresh weight of fruit, asdetermined by the method described herein below. F3 seeds were sown andten plants of each of the F3 plants of these three F2 selections (termedF2-24, F2-82 and F2-134) were grown, and fruit was allowed to remain onthe vine past the normal stage of ripening and harvest. Fruit from theseF3 plants were generally yellow when ripe and did not turn red evenafter the normal ripening stage. Among the F3 plants one plant(F3-203-10, derived from F2-134) showed the characteristic of signs offruit dehydration, evidenced by wrinkling of the fruit skin.

A pedigree breeding program was developed to obtain tomatoes withreduced water content using as a selection system signs of fruitdehydration as evidenced by wrinkling of the fruit skin after the redripe stage. This breeding strategy consisted of selfing F3-203-10 untilthe F4 generation and backcrossing to L. esculentum breeding line L-27 ,with the product of this cross being selfed for four additionalgenerations to produce the BC1F4 population. Lines of this population(lines 901 and 903) as well as hybrid plants derived from crossesbetween this population and commercial tomato cultivars (cv. F139 andcv. BR124) produced plants that all showed the trait of fruitdehydration as evidenced by wrinkling of the ripe fruit skin. Thepresence of the trait in the hybrid plants indicates that the trait isheritable, governed by dominant genetic factors, and can be selected forin the early generations of the breeding program.

EXAMPLE 2

Pollen from one plant (F2-82) which was characterized by high solublesugar level in the mature fruit (71 mg soluble sugar, composed ofsucrose, glucose and fructose, per gram fresh weight of fruit) was usedto pollinate two standard, industry type tomatoes (breeding lines A701and 699) for the production of two backcross-F1 (BC-F1) populations.One-hundred BC-F2 plants from each of the two hybrids were grown and thepresence of signs of fruit dehydration, evidenced by wrinkling of thefruit skin, were seen in fruit of plants from these F2 populations. Thisshows that even at early stages of a selection program, the trait can beselected for without large populations of plants.

Experiment 3

Fruit of progeny of advanced lines derived from the lines described inexperiment 1, that showed the characteristic to dehydrate on the vine,as evidenced by the wrinkling of the fruit were harvested and the juicepressed and Brix of the expressed juice was measured by a digitalrefractometer (Atago model X-1). The following table showscharacteristic Brix values of some of the partially wrinkled but notfully dehydrated. The results of this experiment indicate that the traitof fruit dehydration and increase in Brix value is a selectableinherited trait. The parental selection (self of 1465-3) was partiallydehydrated as was the F1 hybrid between 1465-3 and the cherry cultivarF139. This indicates that the trait is at least partially dominant inits inheritance pattern. Similarly, 3 representative plant selectionsfrom the F2 population (1730) derived from the self of the F1(1465-3×F139) which showed the trait of fruit wrinkling are presentedand indicates that the selection method can be used in the segregatingF2 population.

TABLE 1 Brix values of partially dehydrated tomato fruit, harvested fromthe vine at the stage when fruit wrinkling was visually observable.Fruit size was of the cherry-tomato size (approx. 10–15 gr). tomatoplant cross generation Brix 1630-1 + 2 1465-3 self BC2F2 19.2 1631-11465-3 × cv.139 BC3F1 17.4 1730-3 1631-2 self BC3F2 22.4 1730-4 1631-2self BC3F2 29.0 1730-5 1631-2 self BC3F2 11.1Experiment 4

In an experiment to determine whether the dehydration process can takeplace after removal from the vine, red ripe fruits from a BCF3population were harvested, as above, and allowed to remain and dehydrateon netted screens on the laboratory bench without temperature control.After approximately one month the fruit had reached 86.2% dry weight,and were generally unaccompanied by microbial spoilage. Percent dryweight was calculated as the percentage of weight after drying in aforced air oven at 60° C. for 24 overnight, compared to the weight offruit prior to oven drying. Ten representative fruit were used tocalculate the percent dry weight.

Such fruit has been maintained for over a year at 5° C. and at roomtemperature in an uncontrolled environment for at least 5 months,without further decay. The results of these experiments indicate thatthe dehydrated fruit may be harvested at various stages of dehydration(even before dehydration commences) and that dehydration of the fruitmay also continue after detachment from the vine.

Experiment 5

In order to characterize the development of the dehydration process anexperiment was carried out in which 14 red-ripe fruit from plants whichshowed the trait of dehydration of the fruit, but which themselves hadnot yet reached the dehydration stage, were selected. Seven of thesefruit were harvested when red-ripe and analyzed immediately, asdescribed below. The other seven fruit were allowed to remain attachedto the vine for an additional 14 days and, when fruit wrinkling wasobserved, were analyzed, as follows.

Each fruit was individually weighed, a sample of the fruit juice wastested by refractometer, for Brix value. An additional sample of eachfruit was weighed fresh and then dried in an oven, as described above,for the calculation of percent dry weight. A third portion of each fruitwas used for the analysis of individual soluble sugar levels, asfollows.

Individual fruits were harvested and a portion of the fruit pericarp wasplaced in 80% ethyl alcohol and heated to 70° C. in order to stopenzymatic activity and extract the soluble sugars. Soluble sugars wereextracted three times in successive changes of 80% alcohol which wasthen evaporated.

The sugars were then dissolved in double distilled water, centrifuged at5,000 rpm in an eppendorf centrifuge tube for 15 minutes to remove celldebris and 0.5 ml aliquot passed through a 0.45 micron filter inpreparation for high Pressure Liquid Chromatography (HPLC) analysis.HPLC analysis was performed using a BioRad (Richmond, Calif., USA) FastCarbohydrate column for the separation of glucose, fructose and sucroseaccording to the manufacturer's instructions. The sugars were identifiedand quantified according to chromatographic behavior of standards forthe sugars which were obtained from Sigma (St. Louis, Mo., USA).

The results of this study are shown in Table 2 and show that thewrinkling phenomenon is accompanied by loss of water from the fruit,leading to an increase in % dry weight, an increases in Brix andindividual sugar concentrations. The dry matter per fruit remainsapproximately the same. This indicated that the phenomenon of fruitwrinkling, and the concomitant increase in sugar concentration and indry matter concentration is primarily one of natural dehydration of thefruit, without a concomitant loss of fruit dry matter content.

TABLE 2 Trait Red ripe Wrinkled Fresh weight (g/fruit) 16.63 12.20Percent dry weight 12.53 17.89 Brix 11.51 14.57 Sugars (mg/gm fr w)Total 66.4 86.4 Sucrose 3.8 6.1 Glucose 30.5 38.4 Fructose 32.1 41.9Water content (g/fruit) 14.55 10.02 Dry weight (g/fruit) 2.08 2.18Sugars (g/fruit) 1.10 1.05

In summary, with the methods of the present invention, a tomato fruitcan be obtained characterized by an untreated skin which permitsdehydration of the fruit so as to obtain wrinkling of the skin, whereinthe dehydration is generally unaccompanied by microbial spoilage. Inanother aspect of the invention, a tomato fruit can be obtainedcharacterized by a capability of natural dehydration while on a tomatoplant, natural dehydration being defined as wrinkling of skin of thetomato fruit when the fruit is allowed to remain on the plant after anormal ripe harvest stage, wherein the natural dehydration is generallyunaccompanied by microbial spoilage.

Alternatively, it is noted that the tomato fruit can be treated with asubstance, such as sulfur dioxide, to help retain skin color during andafter dehydration, such as is done with dried fruits such as raisins.

It will be appreciated by persons skilled in the art that the presentinvention is not limited to what has been particularly shown anddescribed herein above. Rather the scope of the present invention isdefined only by the claims which follow:

1. A method of producing tomato fruit capable of natural dehydration comprising: (a) crossing at least one Lycopersicon esculentum plant with a Lycopersicon hirsutum to produce hybrid plants; and subsequently; (b) self-crossing and/or back-crossing said hybrid plants of step (a); and subsequently (c) growing said hybrid plants of step (b) such that the fruit remains on the vine of said hybrid plants past normal red ripe harvest stage; and subsequently (d) screening said hybrid plants of step (c) and isolating plants having fruit exhibiting a wrinkling phenotype, thereby producing tomato fruit capable of natural dehydration.
 2. The method according to claim 1, wherein step (a) is effected by pollinating, collecting the seeds, and growing said hybrid plants.
 3. The method according to claim 1, further comprising harvesting said tomato fruit following fruit wrinkling.
 4. An isolated whole tomato fruit comprising a genome of the Lycopersicon esculentum species, wherein said genome comprises an introgression from Lycopersicon hirsutum, said introgression allowing natural fruit dehydration which results in skin wrinkling of the tomato fruit.
 5. An isolated whole tomato fruit comprising a genome of the Lycopersicon esculentum species, wherein said genome comprises an introgression from Lycopersicon hirsutum, said introgression causing untreated skin wrinkling of the tomato fruit. 